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We studied the inhibition of fetal (γ-nAChR) and adult (ε-nAChR) muscle-type nicotinic acetylcholine receptors by the two new nondepolarizing muscle relaxants (NDMRs) rocuronium and rapacuronium, the metabolite 3-desacetyl rapacuronium (Org 9488), and five other, longer-used NDMRs (pancuronium, vecuronium, mivacurium, d-tubocurarine, and gallamine). Receptors were expressed in Xenopus laevis oocytes by cytoplasmic injection of subunit complementary RNAs. Functional channels were activated with 10 μM acetylcholine, alone or in combination with various concentrations of the NDMRs. Currents were recorded with a whole-cell two-electrode voltage clamp technique. All NDMRs reversibly inhibited acetylcholine-activated currents in a dose-dependent fashion. Potencies of rapacuronium and Org 9488 were not statistically different at either γ-nAChR (half-maximal response = 58.2 and 36.5 nM, respectively) or ε-nAChR (half-maximal response = 80.3 and 97.7 nM, respectively). The rank order of potencies at the ε-nAChR (pancuronium > vecuronium ∼ mivacurium > rocuronium ∼ d-tubocurarine > rapacuronium ∼ Org 9488 > gallamine) correlated highly with the clinical doses needed to produce 50% twitch depression at the adductor pollicis muscle in adults. Neuromuscular blockade by rapacuronium may be enhanced by its metabolite Org 9488. Different drug-receptor affinities of the tested NDMRs contribute to the differences in clinical dose requirements of these drugs needed to achieve appropriate muscle relaxation.

*Department of Anesthesia and Perioperative Care, University of California, San Francisco; †Department of Anesthesia, Kantonsspital, Basel, Switzerland; ‡Department of Biopharmaceutical Sciences, University of California, San Francisco; and §Department of Anesthesia, University of Cologne, Germany

Supported by National Institutes of Health Grant GM-58149 (CSY).

Presented in part at the annual meeting of the American Society of Anesthesiologists, San Francisco, CA, October 14–18, 2000.

Rapacuronium (recently withdrawn from the market after an unacceptably frequent incidence of bronchospasm) and rocuronium are nondepolarizing muscle relaxants (NDMRs) characterized by a fast onset of muscle relaxation and a short to intermediate duration of action (Fig. 1) (1,2). Approximately 7% of rapacuronium is metabolized to the 3-desacetyl metabolite, Org 9488, which also exerts neuromuscular blocking activity, but little is known about its potency (3,4). Org 9488 may accumulate after prolonged administration of rapacuronium, especially in patients with impaired renal function (5).

NDMRs block neuromuscular transmission by binding competitively to the muscle nicotinic acetylcholine receptor (nAChR). Briefly, the nAChR is a ligand-gated ion channel that consists of four different subunits assembled into a pentameric structure to create a central ion-conducting pore (6,7). Two subtypes of the muscle nAChR have been described: the adult form (ε-nAChR) composed of α2βδε subunits and the fetal form (γ-nAChR) containing α2βγδ subunits (8,9). Replacement of the γ by the ε subunit, which occurs as a developmental switch late in gestation and in the early neonatal period, can have clinical importance, because the two subtypes display different electrophysiological and drug-binding properties (10). In addition, pathophysiological states, such as denervation and burns, result in an upregulation of the expression of the γ subunit and therefore may affect the action of NDMRs (11).

The clinical determination of NDMR potency in humans is typically studied by measuring the twitch response of the adductor pollicis muscle after stimulation of the ulnar nerve (12). Because the stimulation of the ulnar nerve is not well tolerated while individuals are awake (13), they are usually anesthetized for these determinations. However, the types of anesthesia administered, e.g., volatile anesthetics or propofol, have their own effects on the neuromuscular junction and therefore may interfere with potency determinations (14). Furthermore, initial twitch depression occurs only when more than approximately 75% of nAChRs are blocked by NDMRs at one binding site (15). These observations suggest that twitch monitoring, although very important for the clinical use of muscle relaxants, does not necessarily reflect molecular interactions at the receptor level.

The aim of our study was to determine directly the potencies of the two muscle relaxants, rocuronium and rapacuronium, and the active metabolite of rapacuronium, Org 9488, in blocking the function of the nAChR expressed heterologously in Xenopus laevis oocytes. Furthermore, we wanted to compare these results with those obtained with other, longer-used muscle relaxants—vecuronium, pancuronium, mivacurium, d-tubocurarine, and gallamine—of which some have been studied previously (16–18). To assess developmental differences in drug action on the neuromuscular junction, both ε-nAChR and γ-nAChR subtypes were studied (17).

Methods

All experimental procedures involving the South African clawed frog (X. laevis) were approved by the Committee on Animal Research of the University of California, San Francisco, and are similar to those previously described (19).

Adult female frogs (Nasco, Fort Atkinson, WI) were maintained in freshwater holding tanks at 19°C–22°C, with a 12-h light/dark cycle. For the removal of unfertilized oocytes, frogs were anesthetized by immersion in a 0.3% solution of 3-aminobenzoic acid ethyl ester (Sigma Chemical Co., St. Louis, MO) at 4°C. Anesthetized frogs were placed supine on ice, and the ovary on one side was removed through a small incision in one quadrant of the lower abdominal wall. The wound was closed as one layer, and after recovery from anesthesia, frogs were returned to their tanks. After removal, ovaries were teased apart with forceps and washed twice in a calcium-free high-magnesium-containing lactated Ringer’s solution (composition, in mM: 82 NaCl, 2 KCl, 5 HEPES, and 20 MgCl2, pH 7.4) followed by collagenase (type D; Boehringer Mannheim, Indianapolis, IN) treatment (2 mg/mL) for 1–2 h at room temperature with constant agitation to remove the follicular cell layer. Oocytes were washed again and transferred into modified Barth’s solution with HEPES (MBSH) [composition, in mM: 88 NaCl, 1 KCl, 10 HEPES, 7 NaHCO3, 1 CaCl2, and 1 Ca(NO3)2, pH 7.0]. Only mature oocytes (Stage V and VI) were selected for injection by visual inspection under the microscope. Within 16 h after removal, oocytes were injected with either diluted mixtures (αβγδ or αβδε) of complementary RNA (cRNA) transcribed from the plasmids or water as control. Aliquots of each cRNA synthesized from plasmids encoding the α, β, γ, and δ subunits were diluted 1:1000 in ribonuclease-free water and mixed in the ratio of 2:1:1:1, respectively. For the ε subunit, a dilution of 1:20 was mixed with 1:1000 dilutions of α, β, and δ subunits to achieve similar levels of functional receptor expression.

Expression plasmids pSPα1, pGEMβ, pSPγ, and pSPδ, encoding complementary DNA coding sequences for mouse muscle nAChR subunits α, β, γ, and δ, respectively, were provided by Drs. John Forsayeth and Zach Hall (Department of Physiology, University of California, San Francisco, CA), and expression plasmid pSPε was provided by Dr. Paul Gardner (Department of Biochemistry, Dartmouth Medical School, Hanover, NH). These plasmids contain an SP6 promoter 5′ to the translation start codon that allows in vitro synthesis of the RNA that directs the translation of each subunit. After cytoplasmic injections of cRNA, oocytes were maintained before the study in MBSH to which 50 mg/mL gentamycin, 2.5 mM sodium pyruvate, 5% heat-inactivated horse serum, and 5 mM theophylline were added.

Electrophysiological experiments were performed 2–5 days after oocyte injection at room temperature (20°C–22°C). Oocytes were placed in a recording chamber with an approximate volume of 25 μl and continuously superfused at 3–5 mL/min with MBSH containing 0.5 μM atropine sulfate. They were impaled with two glass electrodes filled with 3 M KCl (resistance of 0.4–2.5 MΩ). Acetylcholine (Ach)-mediated currents were recorded with a two-electrode voltage clamp with the holding potential set at −60mV (Axoclamp 2A; Axon Instruments, Foster City, CA). Signals were filtered by using an eight-pole low-pass Bessel filter (Frequency Devices, Haverhill, MA) set at a 40-Hz cutoff before sampling at 100 Hz. Resulting signals were digitized and stored on a Power Macintosh 7100 (Apple Computer, Cupertino, CA) by using data acquisition software (MacLab; ADInstruments, Milford, MA).

ACh, atropine, and gallamine were purchased from Sigma. Muscle relaxants were obtained in preparations for clinical use: rocuronium and rapacuronium (Organon Inc., West Orange, NJ), pancuronium (Elkins-Sinn Inc., Cherry Hill, NJ), vecuronium (Baxter Healthcare Corp., Deerfield, IL), and mivacurium and d-tubocurarine (Abbott Laboratories, Chicago, IL). Org 9488 was a gift from Organon Teknika (Boxtel, The Netherlands). All drugs were dissolved in MBSH. Solutions and their dilutions to the experimental concentrations were prepared immediately before the experiments.

Initially, concentration-response curves for ACh were determined for each subtype of the nAChR. For all subsequent experiments, agonist concentration was set at 10 μM ACh. Test solutions containing either ACh alone or in combination with various concentrations of muscle relaxant were applied for 20 s, and the peak current was determined. The control response to ACh alone was repeated after each application of antagonist. The mean value of these two ACh applications was taken as the average control current, to which the antagonist response was compared (percentage inhibition of average control current) by use of the following equation:MATH

To minimize the amount of desensitization during the course of the experiment, a washout of at least 60 s was applied between each drug application. Data were obtained from five to eight oocytes taken from at least two different batches of oocytes.

The concentration-response relations for each muscle relaxant were fitted by nonlinear regression analysis to the four-parameter logistic equation by using GraphPad Prism software 3.0a for Macintosh (GraphPad Software, San Diego, CA), and the inhibitor concentrations for half-maximal response (IC50) and Hill slopes were determined. Drug potencies at each receptor subtype were tested for significant differences by one-way analysis of variance (ANOVA) followed by Tukey’s test; potency differences between receptor subtypes were compared for each drug by using unpaired two-tailed Student’s t-tests with the same software package. P < 0.05 was considered significant. Results are represented as mean ± sd.

Results

Varying concentrations of ACh (0.1–1000 μM) were applied for 20 s to oocytes expressing either the fetal (α2βγδ) or adult (α2βδε) subtype of the muscle nAChR. ACh-elicited concentration-dependent inward currents were observed. Peak currents were determined and the concentration-response data fitted to a logistic equation. The estimated ACh concentrations achieving 50% of the maximal effect (EC50) were similar for both receptor subtypes, with an EC50 of 12 μM for the fetal and 18 μM for the adult receptor (data not shown). In all subsequent experiments, a concentration of 10 μM ACh was used to activate the nAChRs because this concentration produced large and robust signals without significant desensitization after repeated exposures.

Rapacuronium and Org 9488 reversibly inhibited ACh-induced inward currents in a concentration-dependent fashion (Fig. 2). Both drugs had a similar (P > 0.05) concentration-response relationship at the fetal- and adult-type nAChR (Fig. 3), with IC50 values in the nanomolar range. However, both drugs inhibited the fetal subtype of nAChR more potently (P < 0.05) than the adult subtype (Table 1).

All other tested NDMRs also produced reversible, concentration-dependent inhibition of currents elicited by coapplication of 10 μM ACh on either the ε- or γ-nAChR (Fig. 4). In a comparison of the IC50 concentrations, pancuronium, mivacurium, and rocuronium were also more potent on the γ-nAChR, but d-tubocurarine was more potent on the ε-nAChR; no significant differences were found for vecuronium and gallamine (Table 1). The differences in potency between the receptor subtypes were not consistent for the seven tested NDMRs (two-way ANOVA, P > 0.05).

The rank order of potency according to the estimated IC50 concentrations for the ε-nAChR was pancuronium > vecuronium ∼ mivacurium > rocuronium ∼ d-tubocurarine > rapacuronium ∼ Org 9488 > gallamine and was similar for the γ-nAChR (Table 1). A comparison of the estimated IC50 values at the ε-nAChR of the NDMRs tested in this study with clinical ED50 doses (mg/kg) producing a 50% decrease in the force of contraction of the adductor pollicis muscle in adults (20) showed a strong correlation (Pearson correlation coefficient = 0.98;Fig. 5).

Discussion

Determination of the relative potencies of NDMRs and their metabolites at the nAChR is clinically important because active metabolites may contribute substantially to the muscle-blocking effect of the parent compound (3,21). Although pharmacokinetic factors, such as volume of distribution, protein binding, and drug elimination, are important, studying the effects of NDMRs on different nAChR subtypes (adult versus fetal) may help to explain different dosing requirements for adults versus newborns and infants. To directly compare the efficacy of rocuronium and rapacuronium, as well as its active metabolite Org 9488, and several other NDMRs of various structural classes, on the adult and fetal forms of the nAChR, we used the X. laevis oocyte expression system. For most NDMRs analyzed in this study, we found slightly higher affinities to the fetal receptor subtype, suggesting that drug-receptor affinity is affected by the developmental switch of subunit composition. Our results suggest that rapacuronium and Org 9488 have a similar potency at both receptor subtypes. The relative potencies of the tested NDMRs at the ε-nAChR accurately reflect their clinical potencies in adults.

With the oocyte expression system, large numbers of functional receptors are assembled and inserted into the oocyte membrane. This receptor population can be exposed rapidly to various concentrations of specific agonists or antagonists. We had previously established ACh concentration-response relations, demonstrating that the EC50 values did not differ between the adult and fetal nAChR subtypes (22). We used a holding voltage of −60 mV because the antagonistic effects of NDMRs are independent of holding voltages ranging from −100 to −40 mV (16,17). During the course of these experiments, we used an ACh concentration of 10 μM as a standard test concentration. This concentration was close to the EC50 concentration for both receptor subtypes, ensured robust baseline responses, and minimized receptor desensitization due to repetitive ACh application. The use of mouse receptors is justified because of the close sequence homology and functional similarity with human nAChR; however, there could be species differences in response to NDMRs. Although the concentration of agonist used here (ACh 10 μM) may be smaller than the estimated peak concentrations within the neuromuscular junction, our experiments are probably more physiologically meaningful than pure binding assays using desensitized forms of the nAChR. Desensitized nAChRs show different affinities for modulating agents compared with functional receptors (23).

This is the first report of potency determinations for the muscle relaxants rocuronium, rapacuronium, and Org 9488 using a heterologous receptor expression method. We found that rapacuronium and Org 9488 have similar potency in inhibiting each nAChR subtype. This confirms that Org 9488 has muscle-blocking properties, as reported in an earlier clinical study in adults; however, the modeled drug concentrations at the effector site in that study estimated Org 9488 to be approximately 2 to 3 times more potent than the parent compound (3). Pharmacokinetic/pharmacodynamic modeling is based on several assumptions, which might be responsible for the higher estimate of Org 9488 potency. Given that Org 9488 has a similar, or even higher, potency than rapacuronium and an even longer half-life, Org 9488 may be responsible for unexpected enhancement of neuromuscular blockade, especially if rapacuronium is administered over a prolonged period (3,5). This risk of enhanced muscle block is increased in patients with renal failure because the clearance of Org 9488 is reduced by 85% in these patients compared with healthy volunteers (4).

The aminosteroids rocuronium, rapacuronium, Org 9488, and pancuronium, but not vecuronium, showed greater potency on the fetal subtype compared with the adult subtype of nAChR. The higher potency of pancuronium at the γ-AChR confirms our previously reported findings with dimethylphenyl piperazinium as an agonist (18); however, Garland et al. (17) found pancuronium to be more potent at the ε-nAChR. The equipotent effects of vecuronium on both receptor subtypes is in close agreement with earlier studies (17,22). The receptor affinities of the benzoisoquinolines mivacurium and d-tubocurarine were also affected by the developmental switch of subunit composition; mivacurium was more potent at the fetal subtype, but d-tubocurarine was more potent at the adult subtype of the nAChR. Given that NDMRs are generally more potent in neonates than in adults, our findings with d-tubocurarine suggest that it exerts additional effects on the intact neonatal neuromuscular junction to enhance its potency.

Although most NDMRs showed a higher affinity to the fetal subtype in our study, the differences in drug-receptor affinity between the receptor subtypes are not entirely consistent for all compounds. Although we have no experimental data from these studies to explain this finding, we suggest that this reflects a subtle difference in the binding of the molecular structures within the competitive inhibition site in the two receptor subtypes. At the molecular level, the two agonist binding sites for Ach, as well as the antagonist binding sites for NDMRs, are nonequivalent, with one being formed by contributing portions of α and δ subunits and the other by the α subunit and either the ε or γ subunit (24). The two sites have different binding affinities for agonist and must both be occupied by agonist to open the channel. However, only one site needs to be occupied by an antagonist to produce receptor blockade (25). Again, differences in binding affinity may explain the differences in Hill coefficients that we observed between agonist and antagonist.

The findings of Fletcher and Steinbach (16), who used nAChR expressed in quail fibroblasts, established a rank order of affinities for pancuronium, metocurine, and gallamine for both fetal- and adult-type receptors very similar to those reported here. They reported a 100-fold difference in potency between pancuronium and gallamine for ε-AChR, which is in agreement with our findings of a 200-fold difference (IC50(ε-AChR) for pancuronium = 5.5 nM and for gallamine = 995 nM).

Adequate muscle relaxation for clinical purposes, with the structurally different NDMRs tested in this study, requires that they be given in very different doses spanning a range of approximately 2 orders of magnitude (20). The relative potencies of NDMRs on the ε-nAChR expressed in oocytes presented here correlate closely with the ED50 concentrations of these drugs needed for clinical muscle relaxation in humans (20). This rank order of relative potency also reflects the clinical doses necessary for intubation. This finding provides further validation of the oocyte expression system for pharmacodynamic studies of NDMRs on nAChR subtypes. Our data suggest that the differences in drug dosages needed to achieve clinically appropriate muscle relaxation may be largely attributed to the different drug-receptor affinities rather than to other factors, such as drug binding to plasma proteins or membrane permeability.

The authors thank Beth Sampson and Frank Chen for excellent technical assistance and Charles E. McCulloch (Professor of Biostatistics, Department of Epidemiology and Biostatistics, University of California, San Francisco) for statistical advice. Org 9488 was kindly provided by Organon Teknika, The Netherlands.